A cornea includes five layers of a corneal epithelia layer, a Bowman's membrane, a corneal stromal layer, a Descemet's membrane and a corneal endothelial layer provided in this order from the outside. The corneal endothelial layer present in the innermost position among these layers is a single cell layer, and keeps the corneal thickness constant by taking a substance necessary for the cornea from aqueous humor and discharging fluid of the cornea to the aqueous humor, so as to retain the transparency of the cornea. If the number of corneal endothelial cells is reduced, the fluid cannot be sufficiently discharged, which leads to corneal opacity or a corneal endothelial disease such as bullous keratopathy.
In general, human corneal endothelial cell density is approximately 3000 cells/mm2, but it is lowered to approximately 500 cells/mm2 in a patient suffering from a corneal endothelial disease. However, the human corneal endothelial cells are not grown in vivo, and hence, if they are damaged or largely reduced once, the only basic treatment is transplantation.
A refractory corneal endothelial disease such as bullous keratopathy has been conventionally treated by penetrating keratoplasty, but this treatment has problems of absolute shortage of donors and rejection occurring after the transplantation. In order to reduce the rejection, a method in which a corneal endothelium (partly including corneal stromal cells) of an imported human eye bank cornea is collected to be transplanted to a diseased eye (Descement stripping endothelial keratoplasty: DSEK) has been employed, but the problem of shortage of donors still cannot be overcome by the DSEK.
A method in which corneal endothelial cells are grown in vitro to be used for a treatment has been tried (for example, Non-patent Literature 1 and Patent Literatures 1 and 2). In a conventional culture method using a serum, however, corneal endothelial cells are changed in cell shape to lose the corneal endothelial function through long-term culture, and ultimately, the growth is completely stopped, and hence, the cells can be generally grown merely up to a passage number of 5 to 7 (Non-patent Literature 2). This is probably because, in an existing method, corneal endothelial progenitor cells cannot be selectively grown and hence differentiated cells alone are temporarily grown.
It has been reported that a sphere obtained by suspension culture of corneal endothelial cells has properties of a corneal endothelial progenitor cell (Non-patent Literature 3 and Patent Literature 3). The sphere thus obtained does not, however, express p75, a marker for neural crest stem cells, which are an origin of a corneal endothelium, and its undifferentiation is unknown. P75 is expressed in merely stem cells, including neural crest stem cells, present in vivo in a small number, and is one of markers the most reliable as an index of undifferentiation.
As described so far, under the present condition, although there is a technology to grow mature cultured corneal endothelial cells, the transplantation of the cultured corneal endothelial cells is not performed. Therefore, it is desired to develop a technique to obtain and grow corneal endothelial stem cells or corneal endothelial progenitor cells, which can be stably grown to be induced into corneal endothelial cells with good quality.